The high FKBP1A expression in WBCs as a potential screening biomarker for pancreatic cancer.

Given the limitation of current routine approaches for pancreatic cancer screening and detection, the mortality rate of pancreatic cancer cases is still critical. The development of blood-based molecular biomarkers for pancreatic cancer screening and early detection which provide less-invasive, high-sensitivity, and cost-effective, is urgently needed. The goal of this study is to identify and validate the potential molecular biomarkers in white blood cells (WBCs) of pancreatic cancer patients. Gene expression profiles of pancreatic cancer patients from NCBI GEO database were analyzed by CU-DREAM. Then, mRNA expression levels of three candidate genes were determined by quantitative RT-PCR in WBCs of pancreatic cancer patients (N = 27) and healthy controls (N = 51). ROC analysis was performed to assess the performance of each candidate gene. A total of 29 upregulated genes were identified and three selected genes were performed gene expression analysis. Our results revealed high mRNA expression levels in WBCs of pancreatic cancer patients in all selected genes, including FKBP1A (p < 0.0001), PLD1 (p < 0.0001), and PSMA4 (p = 0.0002). Among candidate genes, FKBP1A mRNA expression level was remarkably increased in the pancreatic cancer samples and also in the early stage (p < 0.0001). Moreover, FKBP1A showed the greatest performance to discriminate patients with pancreatic cancer from healthy individuals than other genes with the 88.9% sensitivity, 84.3% specificity, and 90.1% accuracy. Our findings demonstrated that the alteration of FKBP1A gene in WBCs serves as a novel valuable biomarker for patients with pancreatic cancer. Detection of FKBP1A mRNA expression level in circulating WBCs, providing high-sensitive, less-invasive, and cost-effective, is simple and feasible for routine clinical setting that can be applied for pancreatic cancer screening and early detection.

CD99 Expression and Prognostic Impact in Glioblastoma: A Single-Center Cohort Study.

Glioblastoma is the most frequent and aggressive brain tumor in adults. This study aims to evaluate the expression and prognostic impact of CD99, a membrane glycoprotein involved in cellular migration and invasion. In a cohort of patients with glioblastoma treated with surgery, radiotherapy and temozolomide, we retrospectively analyzed tumor expression of CD99 by immunohistochemistry (IHC) and by quantitative real-time polymerase chain reaction (qRT-PCR) for both the wild type (CD99wt) and the truncated (CD99sh) isoforms. The impact on overall survival (OS) was assessed with the Kaplan-Meier method and log-rank test and by multivariable Cox regression. Forty-six patients with glioblastoma entered this study. Immunohistochemical expression of CD99 was present in 83%. Only the CD99wt isoform was detected by qRT-PCR and was significantly correlated with CD99 expression evaluated by IHC (rho = 0.309, p = 0.037). CD99 expression was not associated with OS, regardless of the assessment methodology used (p = 0.61 for qRT-PCR and p = 0.73 for IHC). In an exploratory analysis of The Cancer Genome Atlas, casuistry of glioblastomas CD99 expression was not associated with OS nor with progression-free survival. This study confirms a high expression of CD99 in glioblastoma but does not show any significant impact on survival. Further preclinical studies are needed to define its role as a therapeutic target in glioblastoma.

Investigation of Sensitivity of Rapid Diagnosis Tests in Patients with Suspected Malaria

Objective: Malaria has been eradicated in Türkiye as of 2010, but there are imported cases. In this study, we aimed to compare the diagnostic value of two rapid tests; SD Bioline Malaria Ag Pf/Pan (SD-Pf/Pan) and SD Bioline Malaria Ag Pf/Pv (SD-Pf/Pv) with microscopy and real time-polymerase chain reaction (RT-PCR). Methods: Blood samples were taken from all participants. Thick drop smears were prepared. Thick drop smears were examined for malaria positive/negative distinction under the light microscopy. Then, two rapid diagnostic tests (SD-Pf/Pan and SD-Pf/Pv) were performed. After DNA extraction from blood samples, RT-PCR was typed. The data were evaluated with SPSS 21 program of statistics. Results: A total of 30 cases out of 66 suspected malaria cases were detected as positive with microscopy and RT-PCR. Twenty-seven patients were found positive with both SD-Pf/Pan and SD-Pf/Pv tests. Based on the microscopic results as a reference method, SD-Pf/Pan and SD-Pf/Pv rapid diagnostic tests had a 90% sensitivity, 100% specificity, 100% positive predictive value (PPV), and 92.86% negative predictive value (NPV). Based on the RT-PCR results as a reference method, for detection of P. falciparum, both tests had a 95.65% sensitivity, 100% specificity, 100% PPV, and 88.89% NPV. Moreover, while SD-Pf/Pv had a sensitivity, specificity, PPV, and NPV of 100% in detection of P. vivax; SD-Pf/Pan has a 77.78% sensitivity of, 61.90% specificity of, 46.67% PPV, and 86.67% NPV SD-Pf/Pan for detection of PAN. Conclusion: As a result, high sensitivity and specificity were detected in both kits in the diagnosis of malaria infections caused by P. falciparum and P. vivax. Rapid diagnostic tests can be used safely in diagnosis however the diagnosis should be supported by microscopy and RT-PCR methods when they are applicable.

Utility of an in-house real-time PCR in whole blood samples as a minimally invasive method for early and accurate diagnosis of invasive mould infections.

INTRODUCTION: Invasive mould infections (IMIs) are a leading cause of death in patients with compromised immune systems. Proven invasive mould infection requires detection of a fungus by histopathological analysis of a biopsied specimen, sterile culture, or fungal DNA amplification by PCR in tissue. However, the clinical performance of a PCR assay on blood samples taken from patients suspected of invasive mould disease has not been fully evaluated, particularly for the differential diagnosis of invasive aspergillosis (IA) and invasive Mucormycosis (IM). OBJECTIVES: To assess the diagnostic utility of our previously validated in-house real-time PCR in blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. METHODS: All patients with suspected invasive mould infection were prospectively enrolled from May 2021 to July 2021. Conventional fungal diagnosis was performed using tissue and respiratory samples. In-house PCR was performed on blood samples and its diagnostic performance evaluated. RESULTS: A total of 158 cases of suspected invasive mould infection were enrolled in the study. The sensitivity and specificity of in-house PCR performed on blood samples was found to be 92.5% and 81.4% respectively for diagnosis of probable IA, and 65% and 84.62% respectively for diagnosis of proven and probable IM. It was also able to detect 3 out of 5 cases of possible IM where no other microbiological evidence of IM was obtained. CONCLUSIONS: This assay could be helpful in minimally invasive diagnosis of IMIs for patients in whom invasive sampling is not feasible, especially as a preliminary or screening test. It can help in early diagnosis, anticipating conventional laboratory confirmation by days or weeks. Possible correlation between fungal load and mortality can help in initiating aggressive treatment for patients with high initial fungal load.

Application of benzothiadiazole to Cabernet Gernischt grapes (Vitis vinifera L.) for quality improvement: Effects on aroma metabolism precursors and related genes expression.

Pre-harvest spraying of benzothiadiazole (BTH) can improve the winemaking properties of grapes, especially their aroma compounds and phenolics. Limited research has explored the molecular mechanisms by which BTH influences the accumulation of grape aroma precursors during early grape development. This study investigated the effects and putative molecular mechanisms of applying 0.37 mM BTH through whole-plant spraying on the accumulation of aroma metabolism precursors and gene expression in Cabernet Gernischt grapes during ripening. The results showed that BTH treatment increased the levels of fructose, alanine, aspartate, threonine, myristic acid, myristoleic acid, palmitic acid, ß-cryptoxanthin, norisoprenoids and methoxypyrazines. Contrarily, it decreased the levels of glucose, sucrose, phenylalanine, tyrosine, leucine, valine, glycine, arginine, histidine, total unsaturated fatty acids (particularly linoleic acid), zeaxanthin, lutein, and organic acids. Additionally, BTH upregulated the expression of genes associated with the production and degradation of amino acids, fatty acids, and carotenoids while decreasing the expression of genes involved in the synthesis and degradation of soluble sugars and organic acids. Ten different metabolites, including fumaric acid, were identified as potential biological markers for distinguishing BTH-treated grapes from control grapes. The study demonstrates that BTH treatment had a substantial impact on the concentration and developmental patterns of aroma metabolism precursors. Furthermore, it altered the winemaking characteristics of Cabernet Gernischt grapes by modulating genes associated with the production and breakdown of metabolites.

Unraveling Proto-Oncogene (ErbB2) Expression in Patients With Carcinoma Head of Pancreas and Chronic Pancreatitis Patients: A Case-Control Study.

Background The pre-malignant tendency of the normal, non-affected portion of the pancreas is not as well explored as the multicentricity documented in pancreatic cancer cases. In order to ascertain the expression of inflammatory markers and Erythroblastic Oncogene B (ErbB2) in the non-affected pancreas in patients with pancreatic cancer, a case-control study was carried out. Materials and methods In patients who underwent pancreatoduodenectomy for pancreatic cancer (PC), pro-inflammatory genes and a tumor marker, erythroblastic oncogene 2 (ErbB2) in the epidermal growth factor receptor family were analyzed in the pancreatic tissue at the cut surface of the normal pancreas using qRT-PCR. Twenty patients diagnosed with Chronic pancreatitis (CP) after Frey's surgical procedure were selected, and their pancreatic tissues were analyzed as controls. The HPLC-purified primers were designed using National Center for Biotechnology Information (NCBI) software. The primer's specificity was verified for gene expression analysis using the Basic Local Alignment Search Tool (BLAST). The genes under study were normalized using ß-actin as the housekeeping gene, and the 2-ddct method was used to compute the fold change compared to the control sample. Results Patients with margin-positive were not included. Pro-inflammatory genes (TNF-α, NF-kß, and COX-2) had significantly lower foldchange in PC patients compared to the CP group. The CP control group had higher levels of IL-6 gene expression than the PC group. Patients with pancreatic cancer had a considerably higher expression of the ErbB2 gene than patients with CP. Conclusion The upregulated ErbB2 gene in the unaffected pancreatic tissue of pancreatic cancer patients, when compared to controls, indicates that the remaining pancreas may have the capacity to cause cancer. Proto-oncogene may play a role in the pathophysiologic process in patients with pancreatic cancer.

Assessment of Mucin-Associated Gene Expression Levels on the Ocular Surface.

The ocular surface is covered with a mucus layer. The mucin-associated genes expressed in the ocular surface cells include MUC1, MUC4, MUC5AC, and MUC16. Impression cytology is useful for collecting specimens from the ocular surface, their histological examination, and measuring mucin-associated gene expression levels. The expression of mucin-associated gene levels was assessed by quantitative polymerase chain reaction. The expression levels of these mucin-associated genes are potential biomarkers for ocular surface diseases, including dry eye disease.

Exploring the modulation of MLH1 and MSH2 gene expression in hesperetin-treated breast cancer cells (BT-474).

The major mortality factor for women globally is breast cancer, and current treatments have several adverse effects. Hesperetin (HSP) is a flavone that occurs naturally with anti-tumor capabilities and has been investigated as a potential treatment for cancer. This study aimed to investigate the cytotoxic and anti-malignant potential of HSP on breast cancer cells (BT-474) and normal cells (MCF-10a). The results indicated that HSP has dose-dependent cytotoxicity in BT-474 and MCF-10a cells. The elevated concentration of HSP lowered cell viability and proliferation. The half-maximal inhibitory concentration (IC50) of HSP in BT-474 cancer cells after a 48-h exposure was 279.2 µM/ml, while the IC50 in normal cells was 855.4 µM/ml. The cytotoxicity of HSP was more significant in cancer cell lines than in normal cell lines and this aspect presents a favorable factor in utilizing the drug for the treatment of breast cancer. The apoptotic effect of HSP in BT-474 cells was investigated, and it was found that the higher the concentration of HSP more the cells underwent apoptosis. Furthermore, the highest concentration of HSP led to overexpression of the MLH1 and MSH2 genes in both breast cancer and normal cell lines. Overall, our study suggests that HSP has an anticancer effect on breast cancer cell lines, and the effect is concentration dependent.

An overview of point-of-care testing for infections in critically ill patients.

INTRODUCTION: Molecular diagnostic systems for point-of-care (POC) testing are nowadays routinely used and are part of many labs. Although often intended for bedside use outside of the microbiology lab, there is still room for expansion. AREAS COVERED: This review discusses the two techniques that are currently the most widespread, real-time polymerase-chain reaction (RT-PCR) and loop-mediated isothermal amplification (LAMP). An overview is provided of the various manufacturers and products as well as the evidence and current use in clinical practice. The article further sheds light on some newer techniques, such as CRISPR-based diagnostics and lab-on-a-chip, which are still in development. EXPERT OPINION: With many new platforms and techniques still in the pipeline and their potential currently not yet fully exploited, we expect the use of molecular POC testing to increase in the years to come. However, even when used in hospital - in lab, the main advantages of the tests being fast and easy to perform already provide significant benefits in terms of patient outcome.

Clinical and microbiological efficacy of intra-pocket application of diode laser in grade C periodontitis: a randomized controlled clinical trial.

BACKGROUND: Periodontitis is a microbially induced disease destroying structures anchoring teeth to jaw bones. Although metronidazole in combination with spiramycin is the effective conventional treatment of stage III grade C periodontitis, it has several systemic side effects. Laser therapy is widely used nowadays as an adjunct to scaling and root planing (SRP) to modulate inflammatory host response and eradicate microbes, due to bactericidal and detoxifying effects. Since microbiological analysis is one of the diagnostic methods identifying periodontal risk; our research aimed to investigate the efficacy of intra-pocket application of diode laser (980 nm) versus antibiotic therapy in enhancing clinical and microbiological parameters in stage III grade C periodontitis. METHODS: A randomized controlled clinical trial was conducted on fifty patients with stage III grade C periodontitis, divided equally into two groups. We managed test group by SRP with intra-pocket application of diode laser (980 nm) and the control group by SRP with systemic antibiotic administration (spiramycin and metronidazole). Then, we measured periodontal pocket depth (PPD) and clinical attachment loss (CAL) for both groups, before treatment (baseline), four and twelve weeks after. Moreover, we collected gingival crevicular fluid from both groups at baseline, four and twelve weeks after treatment and analyzed by real-time polymerase chain reaction to detect the relative count of Aggregatibacter actinomycetemcomitans and Porhyromonas gingivalis. RESULTS: Compared to baseline, all assessed clinical and microbiological parameters attested improvement at the end of the study period in each group individually with no significant difference between the two studied groups. Although, at twelve weeks, flare up of bacterial levels was detected with systemic antibiotic administration. CONCLUSION: Laser therapy can be considered as an effective treatment modality in stage III grade C periodontitis, avoiding the systemic antibiotic side effects and solving the recurrence problems due to bacterial resistance by long term usage. TRIAL REGISTRATION: NCT05222737 retrospectively on 03/02/2022, Clinicaltrial.gov.

Analytical evaluation of the automated genotyping system (GenPlex) compared to a traditional real-time PCR assay for the detection of high-risk human papillomaviruses.

The detection of high-risk human papillomaviruses (HPVs) is crucial for early screening and preventing cervical cancer. However, the substantial workload in high-level hospitals or the limited resources in primary-level hospitals hinder widespread testing. To address this issue, we explored a sample-to-answer genotyping system and assessed its performance by comparing it with the traditional real-time polymerase chain reaction (PCR) method conducted manually. Samples randomly selected from those undergoing routine real-time PCR detection were re-analyzed using the fully automatic GenPlex® system. This system identifies 24 types of HPV through a combination of ordinary PCR and microarray-based reverse hybridization. Inconsistent results were confirmed by repeated testing with both methods, and the κ concordance test was employed to evaluate differences between the two methods. A total of 365 samples were randomly selected from 7259 women. According to real-time PCR results, 76 were high-risk HPV negative, and 289 were positive. The GenPlex® system achieved a κ value greater than 0.9 (ranging from 0.920 to 1.000, p < 0.0001) for 14 types of high-risk HPV, except HPV 51 (κ = 0.697, p < 0.0001). However, the inconsistent results in high-risk HPV 51 were revealed to be false positive in real-time PCR by other method. When counting by samples without discriminating the high-risk HPV type, the results of both methods were entirely consistent (κ = 1.000, p < 0.0001). Notably, the GenPlex® system identified more positive cases, with 73 having an HPV type not covered by real-time PCR, and 20 potentially due to low DNA concentration undetectable by the latter. Compared with the routinely used real-time PCR assay, the GenPlex® system demonstrated high consistency. Importantly, the system's advantages in automatic operation and a sealed lab-on-chip format respectively reduce manual work and prevent aerosol pollution. For widespread use of GenPlex® system, formal clinical validation following international criteria should be warranted.

Adaptation and validation of a quantitative vanA/vanB DNA screening assay on a high-throughput PCR system.

Vancomycin resistant enterococci (VRE) are a leading cause of ICU-acquired bloodstream infections in Europe. The bacterial load in enteral colonization may be associated with a higher probability of transmission. Here, we aimed to establish a quantitative vanA/vanB DNA real-time PCR assay on a high-throughput system. Limits of detection (LOD), linear range and precision were determined using serial bacterial dilutions. LOD was 46.9 digital copies (dcp)/ml for vanA and 60.8 dcp/ml for vanB. The assay showed excellent linearity between 4.7 × 101 and 3.5 × 105 dcp/ml (vanA) and 6.7 × 102 and 6.7 × 105 dcp/ml (vanB). Sensitivity was 100% for vanA and vanB, with high positive predictive value (PPV) for vanA (100%), but lower PPV for vanB (34.6%) likely due to the presence of vanB DNA positive anerobic bacteria in rectal swabs. Using the assay on enriched VRE broth vanB PPV increased to 87.2%. Quantification revealed median 2.0 × 104 dcp/ml in PCR positive but VRE culture negative samples and median 9.1 × 104 dcp/ml in VRE culture positive patients (maximum: 107 dcp/ml). The automated vanA/B_UTC assay can be used for vanA/vanB detection and quantification in different diagnostic settings and may support future clinical studies assessing the impact of bacterial load on risk of infection and transmission.

Diagnostic performance of cross-priming amplification-based lateral flow assay (CPA-LFA) and real-time PCR for koi herpesvirus (KHV) detection.

Epizootics of Koi herpesvirus (KHV) cause mass mortality in koi carp (Cyprinus rubrofuscus) and common carp (Cyprinus carpio) worldwide. Rapid and accurate virus detection technology is crucial for preventing pathogen spread and minimizing damage. Although several diagnostic assays have been developed for KHV, the analytical and diagnostic performance of the detection methods has not been evaluated. In this study, we developed and validated the diagnostic performance of two molecular diagnostic assays, cross-priming amplification-based lateral flow assay (CPA-LFA) and TaqMan probe-based real-time polymerase chain reaction (PCR). To detect KHV, primers and probe were designed based on the thymidine kinase (TK) genes. The detection limits of developed CPA-LFA and real-time PCR assays were determined to be 675.69 copies/µL and 8.384 copies/µL, respectively. The diagnostic sensitivity and specificity of the developed assay were determined using fish samples (n = 179). CPA-LFA was found to be 93.67% and 100%, respectively, and real-time PCR was found to be 100% and 100%, respectively. Therefore, the newly developed CPA-LFA and real-time PCR assays accurately and rapidly detect KHV. CPA-LFA is particularly suitable for point-of-care diagnosis because of its simple diagnostic process, and real-time PCR analysis is most suitable for precise diagnosis because it can detect low viral loads.

Effect of two irrigating solutions on antimicrobial activity and clinical and radiographic success after endodontic treatment in primary teeth: a randomized clinical trial.

OBJECTIVES: This study evaluated the antimicrobial activity, clinical and radiographic outcome of pulpectomy in primary teeth using either 1% sodium hypochlorite (NaOCl) or 2% chlorhexidine (CHX) as irrigants. MATERIALS AND METHODS: A randomized double-blind controlled clinical study in which primary teeth were allocated to 1% NaOCl (n = 20) and 2% CHX (n = 20) groups. Microbiological collections were performed before and after irrigation for agar culture and real-time polymerase chain reaction (qPCR). Clinical and radiographic success was assessed at different times. Data were submitted to descriptive analysis, chi-square, Mann-Whitney, and Wilcoxon tests (p  < .05). RESULTS: For 1% NaOCl, the following clinical and radiographic success rates were observed: 7 days (93%/80%); 30 days, 3 and 6 months (100%). For 2% CHX: 7 days (73%/53%); 30 days (93%); 3 months (100%/93%); 6 months (100%) (p > .05). One percent NaOCl and 2% CHX effectively reduced total microorganisms (p < .05) but not mutans streptococci (p > .05). In qPCR analysis, the solutions promoted a reduction of total bacteria and Streptococcus mutans, and no difference was observed between times and groups (p > .05). CONCLUSIONS: One percent NaOCl and 2% CHX were effective for clinical and radiographic success and antimicrobial activity in primary teeth submitted to pulpectomy. CLINICAL RELEVANCE: Studying the antimicrobial activity and clinical and radiographic outcomes of pulpectomy in primary teeth using NaOCl and CHX as irrigants is clinically relevant because it provides information for optimizing treatment protocols and improving the quality of care for pediatric patients. It contributes to evidence-based practice and can potentially lead to better outcomes, reduced complications, and enhanced patient experiences.

Comparison of telomere length in patients with bone marrow failure syndromes and healthy controls.

INTRODUCTION: During normal aging, telomeric DNA is gradually lost in dividing somatic cells, and critically short telomeres lead to replicative senescence, apoptosis, or chromosomal instability. We studied telomere length in bone marrow failure syndromes (BMFS) compared to normal healthy population. METHODS: Peripheral blood was collected from the participants, and genomic DNA was extracted. Relative telomere length was measured using a quantitative polymerase chain reaction. Statistical analysis was performed using SPSS and GraphPad Prism 8.2 software. RESULTS: The median age of normal Indian population was 31 (0-60) years. As expected, telomere length (TL) showed a decline with age and no difference in TL between males and females. The median age of 650 patients with aplastic anemia (AA) was 30 (1-60) years. TL was significantly shorter in patients with AA compared to healthy controls (p < .001). In FA and MDS patients, TL was significantly shorter than age-matched healthy controls (p = .028; p < .001), respectively. There was no difference between the median TL in age-matched AA and FA patients (p = .727). However, patients with MDS had shorter TL than age-matched AA (p = .031). CONCLUSION: TL in BMF syndrome patients was significantly shorter than age-matched healthy controls.

Quantitative Real-Time PCR Method to Evaluate Gene Expression in Zebrafish Embryos.

The fast zebrafish embryonic development offers an opportunity for the study of genes crucial for developmental processes. Several genes associated with human diseases have orthologs in zebrafish. Enhanced comprehension of a gene's function can be achieved by knowing when and where it is expressed. This knowledge also makes it possible to alter a gene-by-gene knockdown in a time- and place-specific manner. Moreover, gene expression analyses contribute greatly to teratogenicity studies in zebrafish embryos. Therefore, the importance of examining the differences between the expressions of these genes has increased day by day. The incorporation of reverse transcription (RT) as the initial step prior to thermal cycling in quantitative real-time polymerase chain reaction (RT-qPCR) has made a significant contribution to RNA research. RT-qPCR is the gold standard and an effective method for gene expression analysis. Quick readout, high sensitivity, reproducibility, as well as high potential throughput, along with reliable quantification, are just a few advantages of RT-qPCR. However, there are drawbacks to its application, such as RNA's inherent variability, impurities during RNA extraction, and variations in reverse transcription and PCR efficiencies. Implementing a precise normalization technique is crucial to account for these inaccuracies. In this chapter, the protocol for gene expression analysis by RT-qPCR for zebrafish embryos is explained.

Evaluating environmental DNA detection of a rare fish in turbid water using field and experimental approaches.

Detection sensitivity of aquatic species using environmental DNA (eDNA) generally decreases in turbid water but is poorly characterized. In this study, eDNA detection targeted delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish associated with turbid water. eDNA sampling in the field was first paired with a trawl survey. Species-specific detection using a Taqman qPCR assay showed concordance between the methods, but a weak eDNA signal. Informed by the results of field sampling, an experiment was designed to assess how turbidity and filtration methods influence detection of a rare target. Water from non-turbid (5 NTU) and turbid (50 NTU) estuarine sites was spiked with small volumes (0.5 and 1 mL) of water from a delta smelt tank to generate low eDNA concentrations. Samples were filtered using four filter types: cartridge filters (pore size 0.45 µm) and 47 mm filters (glass fiber, pore size 1.6 µm and polycarbonate, pore sizes 5 and 10 µm). Prefiltration was also tested as an addition to the filtration protocol for turbid water samples. eDNA copy numbers were analyzed using a censored data method for qPCR data. The assay limits and lack of PCR inhibition indicated an optimized assay. Glass fiber filters yielded the highest detection rates and eDNA copies in non-turbid and turbid water. Prefiltration improved detection in turbid water only when used with cartridge and polycarbonate filters. Statistical analysis identified turbidity as a significant effect on detection probability and eDNA copies detected; filter type and an interaction between filter type and prefilter were significant effects on eDNA copies detected, suggesting that particulate-filter interactions can affect detection sensitivity. Pilot experiments and transparent criteria for positive detection could improve eDNA surveys of rare species in turbid environments.

Expression of mRNA-TLR-5 Gene in Patients with Endometriosis using Real-Time PCR in Tehran, Iran.

BACKGROUND: Endometriosis is one of the common diseases of women, especially in reproductive age, and it is one of the most important causes of infertility in women. The aim of this study was to investigate the level of mRNA-TLR-5 expression in women with endometriosis. METHODS: The present study was performed in Nikan Hospital, Tehran, Iran, in 2021. The samples of endometrial mucosa for the eutopic group and an ovarian endometriotic cyst for the ectopic group were obtained from the patients who underwent laparoscopic surgery at the Fetal Infertility Center and were diagnosed with endometriosis. Normal endometrial samples were also obtained from patients who had no history of infertility and underwent laparoscopic TL surgery for reasons other than endometriosis such as ovarian cysts (control group). After RNA extraction and cDNA synthesis, TLR-5 gene expression was evaluated by the Real-Time PCR method. RESULTS: Based on the results of the comparison of TLR-5 gene expression in all three ectopic, eutopic endometrium, and control groups by Real-Time PCR, it was found that the TLR-5 gene expression is significantly higher in ectopic samples than in the other two groups, but there is a significant difference between two utopic and control groups. CONCLUSION: The increase in TLR-5 expression in the ectopic group can probably be a reason for reducing the apoptosis of cells entered into the peritoneal cavity and creating an environment for the survival and proliferation of these cells.

Molecular monitoring of treatment efficacy in human visceral leishmaniasis.

BACKGROUND: Focused efforts of the visceral leishmaniasis elimination program have led to a drastic decline in cases, and the present challenge is disease monitoring, which this study aimed to assess. METHODS: A Leishmania kinetoplastid-targeted qPCR quantified parasite load at disease presentation, and following treatment completion (n=49); an additional 80 cases were monitored after completion of treatment. RESULTS: The parasite load at disease presentation was 13 461.00 (2560.00-37764.00)/µg gDNA, which upon completion of treatment reduced in 47 of 49 cases to 1(1-1)/µg gDNA, p<0.0001. In 80 cases that presented >2 months post-treatment, their parasite burden similarly decreased to 1(1-1)/µg gDNA except in 6 of 80 cases, which were qPCR positive. CONCLUSION: In 129 cases of visceral leishmaniasis, qPCR by quantification of parasite burden proved effective for monitoring treatment.